2.

a. The liver homogenate serves as a source of mitochondria because they are known to have relatively
higher mitochondria compared to other somatic cells due to its metabolic functions of detoxification.
Since succinate dehydrogenase enzyme is embedded in the mitochondria, the liver cells were used to
observe activity of SDH

b. Cyanide is an irreversible inhibitor that covalently binds to Cytochrome C oxidase and permanently
deforms the component which then eventually stops the reaction pathway.

c. Malonate acts as a competitive inhibitor because it is analogous to substrate succinate. Malonate then
consumes the oxidizing agent, DPIP, that would react with succinate.

d. Water was used to dilute the reagents

The cellular respiration shown by the Succinate Dehydrogenase activity was observed by using
DPIP as an indicator of redox reactions. In this experiment, the liver tissue of a mouse (Mus sp.), which
weighed 1.4 g, was crushed in a tissue homogenizer with trace amounts of 0.05 M phosphate buffer.
The homogenized liver tissue was then transferred to a beaker containing 70 mL of 0.05 M phosphate
buffer. The homogenate was transferred into two 50 mL centrifuge tubes each having 35 mL of the
solution. The tubes were then centrifuged at 7,325 rpm for 10 minutes at 0-4 C. The supernatant was
decanted into chilled centrifuged tubes while the pellet was discarded. The supernatant was further
spun at 3,100 rpm for 15 minutes at the same temperature. The supernatant was collected while the
pellet was discarded. As for the measurement of respiration, 6 tubes were poured with different
reagents. Each tubes had a corresponding blank tube. Tube 1 contained 1 mL each of succinate solution
and distilled water while the blank had 1 mL succinate solution and 2 mL distilled water. Tube 2 was
added with 1 mL each of succinate solution and 0.1 M NaCN while the blank had 1 mL each of succinate
solution, 0.1 M NaCN and distilled water. Tube 3 was poured with 2 mL of distilled water while the blank
has 3 mL. For Tube 4, the contents were 1 mL each of succinate solution and distilled water while the
blank had 1 mL of succinate solution and 2 mL of distilled water. Tube 5 had 1 mL each of succinate
solution and malonate solution while the blank had 1 mL each of succinate solution, distilled water and
malonate solution. Lastly, Tube 6 contained 1 mL of succinate solution plus 0.1 g solid succinate and 1
mL of malonate solution while the blank had 1 mL of succinate solution plus 0.1 g solid succinate, 1 mL
of distilled water and 1 mL of malonate solution. Each tubes and blanks were subjected to the
spectrophotometer at 605 nm for 6 minutes with a 1-minute interval reading. Prior to reading, the blank
of a particular set was added with 1 mL of liver homogenate. While reading the blank, the corresponding
tube was immediately added with 1 mL of DPIP followed by 1 mL of liver homogenate. The timer was
started as the tube was read. As for tube 4, the liver homogenate added was heated.

With regards to cellular respiration observed in the experiment, a normal respiration rate
exhibits a decreasing absorbance as time increases. SDH catalyzes the oxidation of succinate to fumarate
therefore reducing DPIP. The reduced DPIP turns from blue to colorless along with a decrease in
absorbance. The absorbance of the six tubes, which contains the liver homogenate and various
reagents, subjected at 605 nm for 6 minutes with a 1-minute interval reading was shown in Figure 7.1.
Tube 1 (liver homogenate, succinate, DPIP and distilled water) illustrated a decreasing trend because it
does not contain an inhibitor and it has the conditions needed for normal cellular respiration to
proceed. As for Tube 2 (liver homogenate, succinate, DPIP and NaCN) the trend was decreasing initially
but at the latter part the trend was relatively stable. Theoretically, there should be little to no changes
observed because NaCN is an irreversible inhibitor that permanently makes SDH inactive (Giuditta and
Singer 1959). In the initial period the NaCN haven’t bind yet to the active site of the enzyme and as it
binds the activity of the enzyme was blocked. NaCN stopped the electron transport chain resulting to
the accumulation of NADH that inhibited SDH.

Furthermore, Tube 3 (liver homogenate, DPIP and distilled water) also showed a decreasing
absorbance as time increases. Expectedly, there should be no changes observed because of the absence
of succinate. Since the enzyme is SDH, it requires succinate to bind with to cause a reaction. The
deviation from the expected results may be due to the unwashed pipet that was earlier used to transfer
succinate solution. For Tube 4 (heated liver homogenate, succinate, DPIP and distilled water), a
decreasing trend was observed. Hypothetically, the absorbance should show little to no changes
because the heated liver homogenate carried denatured SDH. There was no SDH enzyme that catalyzed
the reaction. The inconsistencies in the data may be caused by insufficient heating of the homogenate
resulting to a partially denatured enzymes.

In Tube 5 (liver homogenate, succinate, malonate and DPIP), the trend initially exhibited a
decreasing trend that eventually stabilizes. Tube 5 had a malonate which is a competitive inhibitor that
binds to the active site of the enzyme. However, it was not dehydrogenated by SDH because of its
different structure, which lacks CH2-CH2 bond in the center of the ion, therefore resulting to a little to
no changes in the absorbance (Chemguide 2016). As aforementioned, the initial decreasing phase may
be attributed to the fact that the malonate has not bind yet to the active site. Lastly, Tube 6 (liver
homogenate, succinate malonate and DPIP) a decreasing trend was shown. The solution had an
increased concentration of succinate to overcome the competitive inhibition caused by the malonate
(Chemguide 2016). A moderate respiration rate was observed as some of the succinate have overcome
the competition with malonate.

In Figure 7.2, the computed average rate of the six tubes was exhibited. From the graph, it was
observed that the Tube 3 has the highest value of respiration rate followed by Tube 1, then Tube 6, Tube
4, Tube 5 and Tube 2. Theoretically, Tube 1 should have the fastest respiration rate because it met the
conditions required for the normal respiration rate and it does not have any inhibitors. Tube 1 should be
followed by Tube 6 since the increased concentration allowed moderate respiration to occur as it
overcomes the inhibition of malonate. Tubes 2, 3, 4 and 5 should have little to no respiration rate
because of the absence of the substrate or the enzyme or the presence of an inhibitor. The deviations
from the trend was attributed to the contamination by the unwashed pipets and also the insufficient
wiping of the cuvettes resulting to an erroneous absorbance.

REFERENCES:

Chemguide (2016). Enzyme inhibitors. Retrieved from

http://www.chemguide.co.uk/organicprops/aminoacids/enzymes3.html on March 9, 2017
Giuditta A. and Singer T.P. (1959). Studies on siccinic dehydrogenase: XI. Inactivation by cyanide and its
mechanism. The Journal of Biological Chemistry, 234:666-671.

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